This research proposal focuses on elucidating the nature of the biomolecular interaction of parathyroid hormone (PTH) with its human receptor (hPTH/PTHrP-Rc) using an approach which integrates chemistry, molecular biology, pharmacology and molecular modeling. The PTH hormone- -receptor system is rich in structural diversity and possesses features providing an unusually strong foundation for structure--activity studies: a broad array of well-characterized hormone analogs (spanning the full spectrum of pharmacologic properties from agonists of enhanced activity to potent pure antagonists) and the convergence of two hormones (PTH and PTHrP) of limited homology on the same receptor. For the most part, PTH analog design has been performed without benefit of knowledge, regarding receptor structure or details of the molecular interaction between hormone and receptor. In the proposed studies, differences in the interaction with receptor of hormone agonists and antagonists will be defined at the level of contact sites between ligand and cloned human receptor. A novel set of radio-labeled PTH analogs incorporating a photoreactive group will be designed, synthesized, and evaluated in a battery of in vitro bioassays. Photoaffinity crosslinking studies will be performed. Radio-labeled fragments of hormone--receptor conjugates will be isolated and characterized. By moving the crosslinking moiety to different positions along the ligand, a map of the binding surfaces of hormone and receptor will be generated. Putative contact sites will be confirmed by making substitutions in the ligand (chemically) or in the receptor (by site-directed mutagenesis). After each round of crosslinking and characterization, a new set of analogs based on the new insights will be designed. In this manner, these novel ligands will be used in an iterative approach to advance understanding of the hormone- receptor interface. Particular focus will be given to elucidating the structural and interactional requirements for signal transduction. Additional emphasis will be placed on utilizing the isolated recombinant N-terminal extracellular domain (N-ECD) of the receptor. Our finding that the N-ECD is functional opens a new direction of investigation which is likely to represent a "breakthrough" for the study of antagonist-receptor interactions. The N-ECD will be used to study binding, crosslinking and conformation both alone and when interacting with ligand. By integrating "photoaffinity scanning", site-directed mutagenesis, conformational analysis and experimentally-based molecular modeling, we will gain novel insights into the binding interface of hormone and receptor and be able to build an experimentally derived model of the spatial arrangement of the major domains (such as N-ECD and transmembrane helix bundles) of this G-protein-coupled receptor. Having demonstrated both the feasibility and power of this approach in the initial period of the grant award, we are now cell-positioned to obtain novel insights into the PTH-receptor system.